Single-copy insertion of transgenes in Caenorhabditis elegans.
At present, transgenes in Caenorhabditis elegans are generated by injecting DNA into the germline. The DNA assembles into a semistable extrachromosomal array composed of many copies of injected DNA. These transgenes are typically overexpressed in somatic cells and silenced in the germline. We have developed a method that inserts a single copy of a transgene into a defined site. Mobilization of a Mos1 transposon generates a double-strand break in noncoding DNA. The break is repaired by copying DNA from an extrachromosomal template into the chromosomal site. Homozygous single-copy insertions can be obtained in less than 2 weeks by injecting approximately 20 worms. We have successfully inserted transgenes as long as 9 kb and verified that single copies are inserted at the targeted site. Single-copy transgenes are expressed at endogenous levels and can be expressed in the female and male germlines.
Frøkjaer-Jensen
Christian
C
Department of Biology and Howard Hughes Medical Institute, University of Utah, 257 South 1400 East, Salt Lake City, Utah 84112, USA.
Davis
M Wayne
MW
Hopkins
Christopher E
CE
Newman
Blake J
BJ
Thummel
Jason M
JM
Olesen
Søren-Peter
SP
Grunnet
Morten
M
Jorgensen
Erik M
EM
D000818:Animals; D030801:Animals, Genetically Modified; D017173:Caenorhabditis elegans / Q000166:cytology / Q000235:genetics*; D004251:DNA Transposable Elements / Q000235:genetics; D005260:Female; D018628:Gene Dosage; D005854:Germ Cells; D049452:Green Fluorescent Proteins / Q000378:metabolism; D007267:Injections; D008297:Male; D016254:Mutagenesis, Insertional / Q000379:methods*; D019076:Transgenes / Q000235:genetics*
D004251:DNA Transposable Elements; D049452:Green Fluorescent Proteins